Culture cell arrest

For monolayer cultures, the cultures are set up the day before BrdU treatment so that the cells will be in exponential growth before the addition of BrdU or the test compound. After BrdU addition the cells are allowed to undergo the equivalent of two cell cycles before cell harvest. A spindle inhibitor such as colchicine or colcemid is introduced for the final 1-2 h of culture to arrest cells in metaphase, after which the cells are harvested and chromosome preparations are made by routine cytogenetic techniques. 

Experimental samples are mainly derived from tissue culture cells, laboratory animals, or human tissues collected from hospitals after surgical biopsies and autopsies. With human and animal tissue specimens, it is important to arrest metabolic processes within 5-10 min of collection in order to preserve mRNAs from degradation by internal enzymatic reactions (26,27). Most hospitals use 10% buffered formalin as a tissue fixative. Subsequently, each tissue slice is trapped in a paraffin block. Series of 4-5-pm-thick sections are cut and mounted on silanated slides. Formalin-fixed archival tissues have been successfully used in in situ PCR and in situ hybridization protocols (28-32). However, the procedure for RNA protection is not always followed. It is often difficult to alter or control the routine procedures of hospitals for the required protection of mRNAs in surgically removed human tissues. 

Although most cultured cell populations come to rest in Gl, i.e. between cell division and DNA synthesis, a proportion of mouse ear epidermal cells are thought to be arrested in G2 (Gelfant, 1959, 1963), although recent evidence casts doubt on these conclusions (Sauerbom et al., 1978). There is some evidence that human embryonic fibroblasts maintained in culture for 48 passages (i.e. in the terminal phase) may arrest in G2 (Maciero-Coelho et al., 1966) but these are so abnormal as to be of no value in studies of G2. As mentioned in 11.6, some tumour cells arrest in G2 on medium exhaustion, but again many metabolic processes are affected and the cells cannot be considered as typical of G2-phase cells. 

In this test, cultured cells are seeded onto slides and the cells, which had been treated with and without metabolic activation for a short time period (e.g. 3 hours). Where negative or equivocal results are obtained, an independent experiment is conducted in which cells are treated for a long time period (e.g. 20 hours) in the absence of metabolic activation alone, and then sampled and examined for chromosome analysis. In both experiments the cells were sampled 20 hours after the start of treatment, as were the concurrent solvent and positive control cultures. Colcemide was added to each culture 2 hours before sampling in order to arrest cell division. Chromosome preparations were made, fixed, stained and examined. However, if clearly positive results were obtained in the first experiment, those from the second assay were not examined. If equivocal or negative results were... 

The model-based dynamic optimisation results that were obtained from a fixed feed composition, same initial condition as the batch culture, and a feeding interval of 6-12 h, suggested an optimal cell cycle-arrest time at 126 h and supplementation with feed from 48 h onwards. The results of three different fed-batch cultures with identical supplementation strategies but various cell cycle-arrest times are shown in Fig.3. The viable cell concentration, Xy, was closely predicted up to about 80 h. However, after lOOh, Xv decreased significantly in all three cultures. The predicted MAb concentration was in accordance with the experimental results with only a slight under-prediction around 80-100 h. Both model predictions and experimental results indicated a small difference in MAb yield when the cultures were arrested at different times. The optimised fed-batch experiments involved a total of 9 shake flask cultures so the... 

Maltese, W. A. Sheridan, K. M. (1987). Isoprenylated proteins in cultured cells subcellular distribution and changes related to altered morphology and growth arrest induced by mevalonate deprivation. J. Cell Physiol. 133,471-481. 

ITSA1 Cultured cells acetylation Bypasses cell-cycle arrest by trichostatin A Unknown... 

G-quadruplex ligands behave as telomerase inhibitors. (A) Long-term treatment of A549 human cell line with a sub-toxic concentration of 12459 (0.04 i.M) leads to the cell-culture growth arrest after several weeks. (B) TRF decrease induced by 12459 in A549 cells. (C) Senescence induced by 12459, revealed by SA-b galactosidase activity at PD 50... 

Stage 14 Drosophila egg chambers (hereafter referred to as oocytes) are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei (purified from either embryos or tissue culture cells) as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins (Maus et al., 1995). Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. Cell-free nuclear lamina disassembly is ATP dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. 

Fig. 7. TAT activity during mitosis and G1. Monolayer cultures of HTC cells were induced with dexamethasone phosphate (Dex), 10-6 M, for 24 hours prior to exposure to 2 x 10-7/1 colcemid. After 12 hours of colcemid treatment, those cells arrested in metaphase were selectively harvested. The adherent interphase cells served as a control population in the experiment in part A. The populations of synchronized cells (mitotic indices greater than 90 percent) were washed by centrifugation (200 x g) at 37°C and resuspended in fresh, prewarmed medium containing neomycin (50 jug/ml) and, when indicated, Dex 10-5 M. These suspension cultures (about 500,000 cells/ml) were maintained at 37°C, and the appropriate drugs were added as Indicated. Aliquots were removed at the intervals indicated and chilled. The cells were washed with neutral buffer prior to being quick-frozen and stored for subsequent determinations. TAT-specIfic activities (nmoles of product/min/mg protein) were normalized to an initial value of 100 because, even though the induction in each case was at least eightfold, the maximally induced levels varied from experiment to experiment. In the experiment depicted in A, the population of synchronized cells was washed free of colcemid at zero time. Dex was also removed from one half of the population (0—0), but in the other half Dex (10-5 M) was present...

Compounds structurally related to podophyllotoxin often have antimitotic activity. They arrest cultured cells at metaphase and bind to purified tubulin preparations. The lactone ring of podophyllotoxin is involved in this binding (MacRae and Towers, 1984). 

Tramontano, W. a., C. M. Hartnett, D. G. Lynn, and L. S. Evans, Relationship between trigonelline concentration and promotion of cell arrest in G2 in cultured roots of Pisum sativum, Phytochemistry, 21, 1201-1206 (1982). 

Chlorpromazine arrests cultured cells in mitosis and disorganises the organised microtubule structure produced by cyclic adenosine monophosphate (Poffenbarger and Fuller 1977). It causes a reduction in the number of microtubules in spinal ganglion cells (Edstrom et al. 1973, Thyberg et al. 1977) and neuroblastoma cells (EdstrOm et al. 1975) in vitro. The micellar form of chlorpromazine interacts preferentially with one site on brain tubulin (Gann et al. 1981). Ghlorpromazine has been shown to bind reversibly to tubulin prepared from mouse brain via two well-resolved processes (Hin-... 

Fig. 1. Effect of kinetin (-1-) and water (X ) on incorporation of [ -P]Pi into phospholipids of cyto-kinin-responsive samples of soybean cells. Cytokinin-responsive samples of soybean cells are suspension cultured cells of soybean (cv Acme) cotyledonary callus brought to mitotic arrest by cyto-kinin-deprivation. Filter-sterilized kinetin solution (1 ml to 50 ml of cell suspension), or water, was supplied at t = 0. At the same time each sample was supplied with p-P]Pi at 74kBq mV Cells were killed in hot /50-propanol at the times shown on the abscissa. The ordinate represents radioactivity in each phospholipid expressed as a percentage of the total radioactivity recovered from each 2-D TLC plate. The total radioactivity recovered from each plate was 15 min. water = 28 Bq, kinetin = 11 Bq 30 min. water = 12 Bq. kinetin = 12 Bq 60 min, water = 29 Bq. kinetin = 27 Bq...

In cultured carrot cells, temporary auxin deprivation induces at least partial synchronization of cell division (Nishi et al. 1977), a response not shown by cultured tobacco cells. Cell arrest during auxin deprivation appears to be in Gj, suggesting that auxin is needed to initiate the S phase of the cycle. 

---